Immunoglobulins devoid of light chains

ABSTRACT

The invention relates to an immunoglobulin having determined antigen specificity, and having a variable fragment which is derived from a so-called heavy-chain immunoglobulin having two heavy polypeptide chains capable of recognizing and binding one or several antigens and which is further naturally devoid of light chains, wherein the immunoglobulin having determined antigen specificity is devoid of CH1 constant region between the variable region and the hinge region and has within its constant region at least part of a constant region of a human antibody.

RELATED APPLICATIONS

This is a division of application Ser. No. 10/751,826, filed Jan. 5,2004, which is a division of application Ser. No. 09/293,769, filed Apr.19, 1999 and issued as U.S. Pat. No. 6,765,087, which is a division ofapplication Ser. No. 08/471,284, filed Jun. 6, 1995 and issued as U.S.Pat. No. 6,005,079, which is a division of application Ser. No.08/106,944, filed Aug. 17, 1993, now abandoned, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates to new isolated immunoglobulins which are devoidof light polypeptide chains. These immunoglobulins do not consist in thedegradation products of immunoglobulins composed of both heavypolypeptide and light polypeptide chains but to the contrary, theinvention defines a new member of the family of the immunoglobulins,especially a new type of molecules capable of being involved in theimmune recognition. Such immunoglobulins can be used for severalpurposes, especially for diagnosis or therapeutical purposes includingprotection against pathological agents or regulation of the expressionor activity of proteins.

Up to now the structure proposed for immunoglobulins consists of afour-chain model referring to the presence of two identical lightpolypeptide chains (light chains) and two identical heavy polypeptidechains (heavy chains) linked together by disulfide bonds to form a y- orT-shaped macromolecules. These chains are composed of a constant regionand a variable region, the constant region being subdivided in severaldomains. The two heavy polypeptide chains are usually linked bydisulphide bounds in a so-called “hinge region” situated between thefirst and second domains of the constant region.

Among the proteins forming the class of the immunoglobulins, most ofthem are antibodies and accordingly present an antigen binding site orseveral antigen binding sites.

According to the four-chain model, the antigen binding site of anantibody is located in the variable domains of each of the heavy andlight chains, and requires the association of the heavy and the lightchains variable domains.

For the definition of these four-chain model immunoglobulins, referenceis made to Roitt. I et al (Immunology-second-Edition Gower MedicalPublishing USA, 1989). Reference is especially made to the partconcerning the definition of the four-chain immunoglobulins, theirpolypeptidic and genetic structures, the definition of their variableand constant regions and the obtention of the fragments produced byenzymatic degradation according to well known techniques.

SUMMARY OF THE INVENTION

The inventors have surprisingly established that different molecules canbe isolated from animals which naturally produce them, which moleculeshave functional properties of immunoglobulins these functions being insome cases related to structural elements which are distinct from thoseinvolved in the function of four-chain immunoglobulins due for instanceto the absence of light chains.

The invention relates to two-chain model immunoglobulins which neithercorrespond to fragments obtained for instance by the degradation inparticular the enzymatic degradation of a natural four-chain modelimmunoglobulin, nor correspond to the expression in host cells, of DNAcoding for the constant or the variable region of a natural four-chainmodel immunoglobulin or a part of these regions, nor correspond toantibodies produced in lymphopaties for example in mice, rats or human.

E. S. Ward et al (1) have described some experiments performed onvariable domains of heavy polypeptide chains (V_(H)) or/and lightpolypeptide chains (V_(K)/F_(v)) to test the ability of these variabledomains, to bind specific antigens. For this purpose, a library of V_(H)genes was prepared from the spleen genomic DNA of mice previouslyimmunized with these specific antigens.

Ward et al have described in their publication that V_(H) domains arerelatively sticky, presumably due to the exposed hydrophobic surfacenormally capped by the V_(κ) or V_(λ) domains. They consequentlyenvisage that it should be possible to design V_(H) domains havingimproved properties and further that V_(H) domains with bindingactivities could serve as the building blocks for making variablefragments (Fv fragments) or complete antibodies.

The invention does not start from the idea that the different fragments(light and heavy chains) and the different domains of these fragments offour-chain model immunoglobulin can be modified to define new orimproved antigen binding sites or a four-chain model immunoglobulin.

The inventors have determined that immunoglobulins can have a differentstructure than the known four-chain model and that such differentimmunoglobulins offer new means for the preparation of diagnosisreagents, therapeutical agents or any other reagent for use in researchor industrial purposes.

Thus the invention provides new immunoglobulins which are capable ofshowing functional properties of four-chain model immunoglobulinsalthough their structure appears to be more appropriate in manycircumstances for their use, their preparation and in some cases fortheir modification. Moreover these molecules can be considered as leadstructures for the modification of other immunoglobulins. The advantageswhich are provided by these immunoglobulins comprise the possibility toprepare them with an increased facility.

The invention accordingly relates to immunoglobulins characterized inthat they comprise two heavy polypeptide chains sufficient for theformation of a complete antigen binding site or several antigen bindingsites, these immunoglobulins being further devoid of light polypeptidechains. In a particular embodiment of the invention, theseimmunoglobulins are further characterized by the fact that they are theproduct of the expression in a prokaryotic or in a eukaryotic host cell,of a DNA or of a cDNA having the sequence of an immunoglobulin devoid oflight chains as obtainable from lymphocytes or other cells of Camelids.

The immunoglobulins of the invention can be obtained for example fromthe sequences which are described in FIG. 7.

The immunoglobulins of the invention, which are devoid of light chainsare such that the variable domains of their heavy chains have propertiesdiffering from those of the four-chain immunoglobulin V_(H). Thevariable domain of a heavy-chain immunoglobulin of the invention has nonormal interaction sites with the V_(L) or with the C_(H) 1 domain whichdo not exist in the heavy chain immunoglobulins. it is hence a novelfragment in many of its properties such as solubility and position ofthe binding site. For clarity reasons we will call it V_(HH) in thistext to distinguish it from the classical V_(H) of four-chainimmunoglobulins.

By “a complete antigen binding site” it is meant according to theinvention, a site which will alone allow the recognition and completebinding of an antigen. This could be verified by any known methodregarding the testing of the binding affinity.

These immunoglobulins which can be prepared by the technique ofrecombinant DNA, or isolated from animals, will be sometimes called“heavy-chain immunoglobulins” in the following pages. In a preferredembodiment of the invention, these immunoglobulins are in a pure form.

In a first embodiment, the immunoglobulins of the invention areobtainable in prokaryotic cells, especially in E. coli cells by aprocess comprising the steps of:

-   -   a) cloning in a pBLUESCRIPT® vector of a DNA or cDNA sequence        coding for the V_(HH) domain of an immunoglobulin devoid of        light chain obtainable for instance from lymphocytes of        Camelids,    -   b) recovering the cloned fragment after amplification using a 5′        primer containing an Xho site and a 3′ primer containing the Spe        site having the following sequence TC TTA ACT AGT GAG GAG ACG        GTG ACC TG (SEQ ID NO:51),    -   c) cloning the-recovered fragment in phase in the immuno PBS        vector after digestion of the vector with Xho and Spe        restriction enzymes,    -   d) transforming host cells, especially E. coli by transfection        with the recombinant immuno PBS vector of step c,    -   e) recovering the expression product of the V_(HH) coding        sequence, for instance by using antibodies raised against the        dromadary V_(HH) domain.

In another embodiment the immunoglobulins are hetero-specificimmunoglobulins obtainable by a process comprising the steps of:

-   -   obtaining a first DNA or cDNA sequence coding for a V_(HH)        domain or part thereof having a determined specificity against a        given antigen and comprised between Xho and Spe sites,    -   obtaining a second DNA or cDNA sequence coding for a V_(HH)        domain or part thereof, having a determined specificity        different from the specificity of the first DNA or cDNA sequence        and comprised between the Spe and EcoRI sites,    -   digesting an immuno PBS vector with EcoRI and XhoI restriction        enzymes,    -   ligating the obtained DNA or cDNA sequences coding for V_(HH)        domains, so that the DNA or cDNA sequences are serially cloned        in the vector,    -   transforming a host cell, especially E. coli cell by        transfection, and recovering the obtained immunoglobulins.

In another embodiment, the immunoglobulins are obtainable by a processcomprising the steps of:

-   -   obtaining a DNA or cDNA sequence coding for a V_(HH) domain or        part thereof, having a determined specific antigen binding site,    -   amplifying the obtained DNA or cDNA, using a 5′ primer        containing an initiation codon and a HindIII site, and a 3′        primer containing a termination codon having a XhoI site,    -   recombining the amplified DNA or cDNA into the HindIII        (position 2650) and XhoI (position 4067) sites of a plasmid        pMM984,    -   transfecting permissive cells especially NB-E cells with the        recombinant plasmid,    -   recovering the obtained products.

Successful expression can be verified with antibodies directed against aregion of a V_(HH) domain, especially by an ELISA assay.

According to another particular embodiment of this process, theimmunoglobulins are cloned in a parvovirus.

In another example these immunoglobulins are obtainable by a processcomprising the further cloning of a second DNA or cDNA sequence havinganother determined antigen binding site, in the pMM984 plasmid.

Such an Immunoglobulin can be further characterized in that it isobtainable by a process wherein the vector is Yep 52 and the transformedrecombinant cell is a yeast especially S. cerevisiae.

A particular immunoglobulin is characterized in that it has a catalyticactivity, especially in that it is directed against an antigen mimickingan activated state of a given substrate. These catalytic antibodies canbe modified at the level of their biding site, by random or directedmutagenesis in order to increase oe modify their catalytic function.Reference may be made to the publication of Lerner et al (TIBS November1987. 427-430) for the general technique for the preparation of suchcatalytic immunoglobulins.

According to a preferred embodiment, the immunoglobulins of theinvention are characterized in that their variable regions contain inposition 45, an amino-acid which is different from leucine, proline orglutamine residue.

Moreover the heavy-chain immunoglobulins are not products characteristicof lymphocytes of animals nor from lymphocytes of a human patientsuffering from lymphopathies. Such immunoglobulins produced inlymphopathies are monoclonal in origin and result from pathogenicmutations at the genomic level. They have apparently no antigen bindingsite.

The two heavy polypeptide chains of these immunoglobulins can be linkedby a hinge region according to the definition of Roitt et al.

In a particular embodiment of the invention, immunoglobulinscorresponding to the above-defined molecules are capable of acting asantibodies.

The antigen binding site(s) of the immunoglobulins of the invention arelocated in the variable region of the heavy chain.

In a particular group of these immunoglobulins each heavy polypeptidechain contains one antigen binding site on its variable region, andthese sites correspond to the same amino-acid sequence.

In a further embodiment of the invention the immunoglobulins arecharacterized in that their heavy polypeptide chains contain a variableregion (V_(HH)) and a constant region (C_(H)) according to thedefinition of Roitt et al, but are devoid of the first domain of theirconstant region. This first domain of the constant region is calledC_(H) 1.

These immunoglobulins having no C_(H) 1 domain are such that thevariable region of their chains is directly linked to the hinge regionat the C-terminal part of the variable region.

The immunoglobulins of the type described here-above can comprise type Gimmunoglobulins and especially immunoglobulins which are defined asimmunoglobulins of class 2 (IgG2) or immunoglobulins of class 3 (IgG3).

The absence of the light chain and of the first constant domain lead toa modification of the nomenclature of the immunoglobulin fragmentsobtained by enzymatic digestion, according to Roitt et al.

The terms Fc and pFc on the one hand, Fc′ and pFc′ on the other handcorresponding respectively to the papain and pepsin digestion fragmentsare maintained.

The terms Fab F(ab)₂ F(ab!)₂ Fabc, Fd and Fv are no longer applicable intheir original sense as these fragments have either a light chain, thevariable part of the light chain or the C_(H) 1 domain.

The fragments obtained by papain digestion and composed of the V_(HH)domain and the hinge region will be called F V_(HH)h or F(V_(HH)h)₂depending upon whether or not they remain linked by the disulphidebonds.

In another embodiment of the invention, immunoglobulins replying to thehereabove given definitions can be originating from animals especiallyfrom animals of the camelid family. The inventors have found out thatthe heavy-chain immunoglobulins which are present in camelids are notassociated with a pathological situation which would induce theproduction of abnormal antibodies with respect to the four-chainimmunoglobulins. On the basis of a comparative study of old worldcamelids (Camelus bactrianus and Camelus dromaderius) and new worldcamelids (for example Lama Paccos, Lama Glama, and Lama Vicugna) theinventors have shown that the immunoglobulins of the invention, whichare devoid of light polypeptide chains are found in all species.Nevertheless differences may be apparent in molecular weight of theseimmunoglobulins depending on the animals. Especially the molecularweight of a heavy chain contained in these immunoglobulins can be fromapproximately 43 kd to approximately 47 kd, in particular 45 kd.

Advantageously the heavy-chain immunoglobulins of the invention aresecreted in blood of camelids.

Immunoglobulins according to this particular embodiment of the inventionare obtainable by purification from serum of camelids and a process forthe purification is described in details in the examples. In the casewhere the immunoglobulins are obtained from Camelids, the inventionrelates to immunoglobulins which are not in their natural biologicalenvironment.

According to the invention immunoglobulin IgG2 as obtainable bypurification from the serum of camelids can be characterized in that:

-   -   it is not adsorbed by chromatography on Protein G SEPHAROSE        column,    -   it is adsorbed by chromatography on Protein A SEPHAROSE column,    -   it has a molecular weight of around 100 kd after elution with a        pH 4.5 buffer (0.15 M NaCl, 0.58% acetic acid adjusted to pH 4.5        by NaOH),    -   it consists of heavy γ2 polypeptide chains of a molecular weight        of around 46 kd preferably 45 after reduction.

According to a further embodiment of the invention another group ofimmunoglobulins corresponding to IgG3, as obtainable by purificationfrom the serum of Camelids is characterized in that the immunoglobulin:

-   -   is adsorbed by chromatography on a Protein A SEPHAROSE column,        has a molecular weight of around 100 kd after elution with a pH        3.5 buffer (0.15 M NaCl, 0.58% acetic acid),    -   is adsorbed by chromatography on a Protein G SEPHAROSE column        and eluted with pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid),    -   consists of heavy γ3 polypeptide chains of a molecular weight of        around 45 Kd in particular between 43 and 47 kd after reduction.

The immunoglobulins of the invention which are devoid of light chains,nevertheless comprise on their heavy chains a constant region and avariable region. The constant region comprises different domains.

The variable region of immunoglobulins of the invention comprisesframeworks (FW) and complementarity determining regions (CDR),especially 4 frameworks and 3 complementarity regions. It isdistinguished from the four-chain immunoglobulins especially by the factthat this variable region can itself contain an antigen binding site orseveral, without contribution of the variable region of a light chainwhich is absent.

The amino-acid sequences of frameworks 1 and 4 comprise among othersrespectively amino-acid sequences which can be selected from thefollowing: for the framework 1 domain GGSVQTGGSLRLSCEISGLTFD (SEQ IDNO:1) GGSVQTGGSLRLSCAVSGFSFS (SEQ ID NO:2) GGSEQGGGSLRLSCAISGYTYG (SEQID NO:3) GGSVQPGGSLTLSCTVSGATYS (SEQ ID NO:4) CGSVQAGGSLRLSCTGSGFPYS(SEQ ID NO:5) GGSVQAGGSLRLSCVAGFGTS (SEQ ID NO:6) GGSVQAGGSLRLSCVSFSPSS(SEQ ID NO:7) for the framework 4 domain WGQGTQVTVSS (SEQ ID NG:8)WGQGTLVTVSS (SEQ ID NO:9) WGQGAQVTVSS (SEQ ID NO:10) WGQGTQVTASS (SEQ IDNO:11) RGQGTQVTVSL (SEQ ID NO:12) for the CDR3 domain A L Q P G G Y C GY G X - - - - - - - - - - C L (SEQ ID NO:62) V S L M D R I S QH - - - - - - - - - - - - G C (SEQ ID NO:63) V P A H L G P G A I L D L KK Y - - - - - - K Y (SEQ ID NO:64) F C Y S T A G D G G S GE - - - - - - - - - M Y (SEQ ID NO:65) E L S G G S C E L P L LF - - - - - - - - - D Y (SEQ ID NO:66) D W K Y W T C G A Q T G G YF - - - - - - - G Q (SEQ ID NO:67) R L T E M G A C D A R W A T L A T R TF A Y N Y (SEQ ID NO:68) Q K K D R T R W A E P R E W - - - - - - - - N N(SEQ ID NO:69) G S R F S S P V G S T S R L E S - S D Y - - N Y (SEQ IDNO:70) A D P S I Y Y S I L X I E Y - - - - - - - - K Y (SEQ ID NO:71) DS P C Y M P T M P A P P I R D S F G W - - D D (SEQ ID NO:72) T S S F Y WY C T T A P Y - - - - - - - - - N V (SEQ ID NO:73) T E I E W Y G C N L RT T F - - - - - - - - T R (SEQ ID NO:74) N Q L A G G W Y L D P N Y W L SV G A Y - - A I (SEQ ID NO:75) R L T E M G A C D A R W A T L A T R T F AY N Y (SEQ ID NO:76) D G W T R K E G G I G L P W S V Q C E D G Y N Y(SEQ ID NO:77) D S Y P C H L L - - - - - - - - - - - - - - D V (SEQ IDNO:78) V E Y P I A D M C S - - - - - - - - - - - - R Y (SEQ ID NO:79)

As stated above, the immunoglobulins of the invention are preferablydevoid of the totality of their C_(H) 1 domain.

Such immunoglobulins comprise C_(H) 2 and C_(H) 3 domains in theC-terminal region with respect to the hinge region.

According to a particular embodiment of the invention the constantregion of the immunoglobulins comprises C_(H) 2 and C_(H) 3 domainscomprising an amino-acid sequence selected from the following: for theC_(H)2 domain: for the C_(H)2 domain: APELLGGPTVFIFPPKPKDVLSITLTP (SEQID NO:31) APELPGGPSVFVFPTKPKDVLSISGRP (SEQ ID NO:32)APELPGGPSVFVFPPKPKDVLSISGRP (SEQ ID NO:33) APELLGGPSVFIFPPKPKDVLSISGRP(SEQ ID NO:34) for the C_(H)3 domain: GQTREPQVYTLA (SEQ ID NO:35)GQTREPQVYTLAPXRLEL (SEQ ID NO:36) GQPREPQVYTLPPSRDEL (SEQ ID NO:109)GQPREPQVYTLPPSREEM (SEQ ID NO:110) GQPREPQVYTLPPSQEEM (SEQ ID NO:111)

Interestingly the inventors have shown that the hinge region of theimmunoglobulins of the invention can present variable lengths. Whenthese immunoglobulins act as antibodies, the length of the hinge regionwill participate to the determination of the distance separating theantigen binding sites.

Preferably an immunoglobulin according to the invention is characterizedin that its hinge region comprises from 0 to 50 amino-acids.

Particular sequences of hinge region of the immunoglobulins of theinvention are the following. GTNEVCKCPKCP (SEQ ID NO:37) or,EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP (SEQ ID NO:38)

The short hinge region corresponds to an IgG3 molecule and the longhinge sequence corresponds to an IgG2 molecule.

Isolated V_(HH) derived from heavy chain immunoglobulins or V_(HH)libraries corresponding to the heavy chain immunoglobulins can bedistinguished from V_(HH) cloning of four-chain model immunoglobulins onthe basis of sequence features characterizing heavy chainimmunoglobulins.

The camel heavy-chain immunoglobulin V_(HH) region shows a number ofdifferences with the V_(HH) regions derived from 4-chain immunoglobulinsfrom all species examined. At the levels of the residues involved in theV_(HH)/V_(L) interactions, an important difference is noted at the levelof position 45 (FW) which is practically always leucine in the 4-chainimmunoglobulins (98%), the other amino acids at this position beingproline (1%) or glutamine (1%).

In the camel heavy-chain immunoglobulin, in the sequences examined atpresent, leucine at position 45 is only found once. It could originatefrom a four-chain immunoglobulin. In the other cases, it is replaced byarginine, cysteine or glutamic acid residue. The presence of chargedamino acids at this position should contribute to making the V_(HH) moresoluble.

The replacement by camelid specific residues such as those of position45 appears to be interesting for the construction of engineered V_(HH)regions derived from the V_(HH) repertoire of 4-chain immunoglobulins.

A second feature specific of the camelid V_(HH) domain is the frequentpresence of a cysteine in the CDR₃ region associated with a cysteine inthe CDR₁ position 31 or 33 or FW₂ region at position 45. The possibilityof establishing a disulphide bond between the CDR₃ region and the restof the variable domain would contribute to the stability and positioningof the binding site.

With the exception of a single pathogenic myeloma protein (DAW) such adisulphide bond has never been encountered in immunoglobulin V regionsderived from 4 chain immunoglobulins.

The heavy-chain immunoglobulins of the invention have further theparticular advantage of being not sticky. Accordingly theseimmunoglobulins being present in the serum, aggregate much less thanisolated heavy chains of a four-chain immunoglobulins. Theimmunoglobulins of the invention are soluble to a concentration above0.5 mg/ml, preferably above 1 mg/ml and more advantageously above 2mg/ml.

These immunoglobulins further bear an extensive antigen bindingrepertoire and undergo affinity and specificity maturation in vivo.Accordingly they allow the isolation and the preparation of antibodieshaving defined specificity, regarding determined antigens.

Another interesting property of the immunoglobulins of the invention isthat they can be modified and especially humanized. Especially it ispossible to replace all or part of the constant region of theseimmunoglobulins by all or part of a constant region of a human antibody.For example the C_(H)2 and/or C_(H)3 domains of the immunoglobulin couldbe replaced by the C_(H)2 and/or C_(H)3 domains of the IgG γ3 humanimmunoglobulin.

In such humanized antibodies it is also possible to replace a part ofthe variable sequence, namely one or more of the framework residueswhich do not intervene in the binding site by human framework residues,or by a part of a human antibody.

Conversely features (especially peptide fragments) of heavy-chainimmunoglobulin V_(HH) regions, could be introduced into the V_(H) orV_(L) regions derived from four-chain immunoglobulins with for instancethe aim of achieving greater solubility of the immunoglobulins.

The invention further relates to a fragment of an immunoglobulin whichhas been described hereabove and especially to a fragment selected fromthe following group:

-   -   a fragment corresponding to one heavy polypeptide chain of an        immunoglobulin devoid of light chains,    -   fragments obtained by enzymatic digestion of the immunoglobulins        of the invention, especially those obtained by partial digestion        with papain leading to the Fc fragment (constant fragment) and        leading to FV_(HH)h fragment (containing the antigen binding        sites of the heavy chains) or its dimer F(V_(HH)h)₂, or a        fragment obtained by further digestion with papain of the Fc        fragment, leading to the pFc fragment corresponding to the        C-terminal part of the Fc fragment,    -   homologous fragments obtained with other proteolytic enzymes,    -   a fragment of at least 10 preferably 20 amino acids of the        variable region of the immunoglobulin, or the complete variable        region, especially a fragment corresponding to the isolated        V_(HH) domains or to the V_(HH) dimers linked to the hinge        disulphide,    -   a fragment corresponding to the hinge region of the        immunoglobulin, or to at least 6 amino acids of this hinge        region,    -   a fragment of the hinge region comprising a repeated sequence of        Pro-X,    -   a fragment corresponding to at least 10 preferably 20 amino        acids of the constant region or to the complete constant region        of the immunoglobulin.

The invention also relates to a fragment comprising a repeated sequence,Pro-X which repeated sequence contains at least 3 repeats of Pro-X, Xbeing any amino-acid and preferably Gln (glutamine), Lys (lysine) or Glu(acide glutamique); a particular repeated fragment is composed of a12-fold repeat of the sequence Pro-X.

Such a fragment can be advantageously used as a link between differenttypes of molecules.

The amino-acids of the Pro-X sequence are chosen among any natural ornon natural amino-acids.

The fragments can be obtained by enzymatic degradation of theimmunoglobulins. They can also be obtained by expression in cells ororganisms, of nucleotide sequence coding for the immunoglobulins, orthey can be chemically synthesized.

The invention also relates to anti-idiotypes antibodies belonging to theheavy chain immunoglobulin classes. Such anti-idiotypes can be producedagainst human or animal idiotypes. A property of these anti-idiotypes isthat they can be used as idiotypic vaccines, in particular forvaccination against glycoproteins or glycolipids and where thecarbohydrate determines the epitope.

The invention also relates to anti-idiotypes capable of recognizingidiotypes of heavy-chain immunoglobulins.

Such anti-idiotype antibodies can be either syngeneic antibodies orallogenic or xenogeneic antibodies.

The invention also concerns nucleotide sequences coding for all or partof a protein which amino-acid sequence comprises a peptide sequenceselected from the following: G G S V Q T G G S L R L S C E I S G L T F D(SEQ ID NO:1) G G S V Q T G G S L R L S C A V S G F S F S (SEQ ID NO:2)G G S E Q G G G S L R L S C A I S G Y T Y G (SEQ ID NO:3) G G S V Q P GG S L T L S C T V S G A T Y S (SEQ ID NO:4) C G S V Q A G G S L R L S CT G S G F P Y S (SEQ ID NO:5) G G S V Q A G G S L R L S C V A G F G T S(SEQ ID NO:6) G G S V Q A G G S L R L S C V S F S P S S (SEQ ID NO:7) WG Q G T Q V T V S S (SEQ ID NO:8) W G Q G T L V T V S S (SEQ ID NO:9) WG Q G A Q V T V S S (SEQ ID NO:10) W G Q G T Q V T A S S (SEQ ID NO:11)R G Q G T Q V T V S L (SEQ ID NO:12) A L Q P G G Y C G Y GX - - - - - - - - - - C L (SEQ ID NO:62) V S L M D R I S QH - - - - - - - - - - - - G C (SEQ ID NO:63) V P A H L G P G A I L D L KK Y - - - - - - K Y (SEQ ID NO:64) F C Y S T A G D G G S GE - - - - - - - - - M Y (SEQ ID NO:65) E L S G G S C E L P L LF - - - - - - - - - D Y (SEQ ID NO:66) D W K Y W T C G A Q T G G YF - - - - - - - G Q (SEQ ID NO:67) R L T E M G A C D A R W A T L A T R TF A Y N Y (SEQ ID NO:68) Q K K D R T R W A E P R E W - - - - - - - - N N(SEQ ID NO:69) G S R F S S P V G S T S R L E S - S D Y - - N Y (SEQ IDNO:70) A D P S I Y Y S I L X I E Y - - - - - - - - K Y (SEQ ID NO:71) DS P C Y M P T M P A P P I R D S F G W - - D D (SEQ ID NO:72) T S S F Y WY C T T A P Y - - - - - - - - - N V (SEQ ID NO:73) T E I E W Y G C N L RT T F - - - - - - - - T R (SEQ ID NO:74) N Q L A G G W Y L D P N Y W L SV G A Y - - A I (SEQ ID NO:75) R L T E M G A C D A R W A T L A T R T F AY N Y (SEQ ID NO:76) D G W T R K E G G I G L P W S V Q C E D G Y N Y(SEQ ID NO:77) D S Y P C H L L - - - - - - - - - - - - - - D V (SEQ IDNO:78) V E Y P I A D M C S - - - - - - - - - - - - R Y (SEQ ID NO:79)APELLGGPSVFVFPPKPKDVLSISGXPK (SEQ ID NO:39) APELPGGPSVFVFPTKPKDVLSISGRPK(SEQ ID NO:40) APELPGGPSVFVFPPKPKDVLSISGRPK (SEQ ID NO:41)APELLGGPSVFIFPPKPKDVLSISGRPK (SEQ ID NO:42) GQTREPQVYTLAPXRLEL (SEQ IDNO:36) GQPREPQVYTLPPSRDEL (SEQ ID NO:109) GQPREPQVYTLPPSREEM (SEQ IDNO:110) GQPREPQVYTLPPSQEEM (SEQ ID NO:111)VTVSSGTNEVCKCPKCPAPELPGGPSVFVFP (SEQ ID NO:43) or,VTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPAPELLGGPSVFIFP (SEQ ID NO:44)GTNEVCKCPKCP (SEQ ID NO:37) APELPGGPSVFVFP (SEQ ID NO:45)EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP (SEQ ID NO:38) APELLGGPSVFIFP. (SEQID NO:46)

Such nucleotide sequences can be deduced from the amino-acid sequencestaking into account the deneneracy of the genetic code. They can besynthesized or isolated from cells producing immunoglobulins of theinvention.

A procedure for the obtention of such DNA sequences is described in theexamples.

The invention also contemplates RNA, especially mRNA sequencescorresponding to these DNA sequences, and also corresponding cDNAsequences.

The nucleotide sequences of the invention can further be used for thepreparation of primers appropriate for the detection in cells orscreening of DNA or cDNA libraries to isolate nucleotide sequencescoding for immunoglobulins of the invention.

Such nucleotide sequences can be used for the preparation of recombinantvectors and the expression of these sequences contained in the vectorsby host cells especially prokaryotic cells like bacteria or alsoeukaryotic cells and for example CHO cells, insect cells, simian cellslike Vero cells, or any other mammalian cells. Especially the fact thatthe immunoglobulins of he invention are devoid of light chains permitsto secrete them in eukaryotic cells since there is no need to haverecourse to the step consisting in the formation of the BIP proteinwhich is required in the four-chain immunoglobulins.

The inadequacies of the known methods for producing monoclonalantibodies or immunoglobulins by recombinant DNA technology comes fromthe necessity in the vast majority of cases to clone simultaneously theV_(H) and V_(L) domains corresponding to the specific binding site of 4chain immunoglobulins. The animals and especially camelids which produceheavy-chain immunoglobulins according to the invention, and possiblyother vertebrate species are capable of producing heavy-chainimmunoglobulins of which the binding site is located exclusively in theV_(HH) domain. Unlike the few heavy-chain immunoglobulins produced inother species by chain separation or by direct cloning, the camelidheavy-chain immunoglobulins have undergone extensive maturation in vivo.Moreover their V region has naturally evolved to function in absence ofthe V_(L). They are therefore ideal for producing monoclonal antibodiesby recombinant DNA technology. As the obtention of specific antigenbinding clones does not depend on a stochastic process necessitating avery large number of recombinant cells, this allows also a much moreextensive examination of the repertoire.

This can be done at the level of the non rearranged V_(HH) repertoireusing DNA derived from an arbitrarily chosen tissue or cell type or atthe level of the rearranged V_(HH) repertoire, using DNA obtained from Blymphocytes. More interesting however is to transcribe the mRNA fromantibody producing cells and to clone the cDNA with or without prioramplification into an adequate vector. This will result in the obtentionof antibodies which have already undergone affinity maturation.

The examination of a large repertoire should prove to be particularlyuseful in the search for antibodies with catalytic activities.

The invention thus provides libraries which can be generated in a waywhich includes part of the hinge sequence, the identification is simpleas the hinge is directly attached to the V_(HH) domain.

These libraries can be obtained by cloning cDNA from lymphoid cells withor without prior PCR amplification. The PCR primers are located in thepromoter, leader or framework sequences of the V_(HH) for the 5′ primerand in the hinge, C_(H)2, C_(H)3, 3′ untranslated region or polyA tailfor the 3′ primer. A size selection of amplified material allows theconstruction of a library limited to heavy chain immunoglobulins.

In a particular example, the following 3′ primer in which a KpnI sitehas been constructed and which corresponds to amino-acids 313 to 319(CGC CAT CAA GGT AAC AGT TGA) (SEQ ID NO:47) is used in conjunction withmouse V_(HH) primers described by Sestry et al and containing a Xho siteAG GTC CAG CTG CTC GAG TCT GG (SEQ ID NO:48) AG CTC CAG CTG CTC GAG TCTGG (SEQ ID NO:49) AG GTC CAG CTT CTC GAG TCT GG (SEQ ID NO:50)               XhoI site

These primers yield a library of camelid heavy chain immunoglobulinscomprising the V_(HH) region (related to mouse or human subgroup III),the hinge and a section of C_(H)2.

In another example, the cDNA is polyadenylated at its 5′ end and themouse specific V_(HH) primers are replaced by a poly T primer with aninbuilt XhoI site, at the level of nucleotide 12.

CTCGAGT₂.

The same 3′ primer with a KpnI site is used.

This method generates a library containing all subgroups ofimmunoglobulins.

Part of the interest in cloning a region encompassing the hinge-C_(H)2link is that in both γ2 and γ3, a Sac site is present immediately afterthe hinge. This site allows the grafting of the sequence coding for theV_(HH) and the hinge onto the Fc region of other immunoglobulins, inparticular the human IgG₁ and IgG₃ which have the same amino acidsequence at this site (Glu₂₄₆ Leu₂₄₇).

As an example, the invention contemplates a cDNA library composed ofnucleotide sequences coding for a heavy-chain immunoglobulin, such asobtained by performing the following steps:

a) treating a sample containing lymphoid cells, especially periferal,lymphocytes, spleen cells, lymph nodes or another lyphoid tissue from ahealthy animal, especially selected among the Camelids, in order toseparate the lymphoid cells,

b) separating polyadenylated RNA from the other nucleic acids andcomponents of the cells,

c) reacting the obtained RNA with a reverse transcriptase in order toobtain the corresponding cDNA,

d) contacting the cDNA of step c) with 5′ primers corresponding to mouseV_(H) domain of four-chain immunoglobulins, which primer contains adetermined restriction site, for example an XhoI site and with 3′primers corresponding to the N-terminal part of a C_(H)2 domaincontaining a KpnI site,

e) amplifying the DNA, f) cloning the amplified sequence in a vector,especially in a BLUESCRIPT® vector,

g) recovering the clones hybridizing with a probe corresponding to thesequence coding for a constant domain from an isolated heavy-chainimmunoglobulin.

This cloning gives rise to clones containing DNA sequences including thesequence coding for the hinge. It thus permits the characterization ofthe subclass of the immunoglobulin and the SacI site useful for graftingthe FV_(HH)h to the Fc region.

The recovery of the sequences coding for the heavy-chain immunoglobulinscan also be achieved by the selection of clones containing DNA sequenceshaving a size compatible with the lack of the C_(H)1 domain.

It is possible according to another embodiment of the invention, to addthe following steps between steps c) and d) of the above process:

in the presence of a DNA polymerase and of deoxyribonucleotidetriphosphates, contacting said cDNA with oligonucleotide degeneratedprimers, which sequences are capable of coding for the hinge region andN-terminal V_(HH) domain of an immunoglobulin, the primers being capableof hybridizing with the cDNA and capable of initiating the extension ofa DNA sequence complementary to the cDNA used as template,

recovering the amplified DNA.

The clones can be expressed in several types of expression vectors. Asan example using a commercially available vector Immuno PBS (Huse et al:Science (1989) 246, 1275), clones produced in pBLUESCRIPT® according tothe above described procedure, are recovered by PCR using the same XhoIcontaining 5′ primer and a new 3′ primer, corresponding to residues113-103 in the framework of the immunoglobulins, in which an Spe sitehas been constructed: TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ IDNO:51). This procedure allows the cloning of the V_(HH) in the Xho/Spesite of the Immuno PBS vector. However, the 3′ end of the gene is not inphase with the identification “tag” and the stop codon of the vector. Toachieve this, the construct is cut with Spe and the 4 base overhangs arefilled in, using the Klenow fragment after which the vector isreligated. A further refinement consists in replacing the marker (“tag”)with a poly histidine so that metal purification of the cloned V_(HH)can be performed. To achieve this a Spe/EcoRI double strandedoligo-nucleotide coding for 6 histidines and a termination codon isfirst constructed by synthesis of both strands followed by heating andannealing: CTA GTG CAC CAC CAT CAC CAT CAC TAA* TAG* (SEQ ID NO:52)     AC GTG GTG GTA GTG GTA GTG ATT ATC TTA A (SEQ ID NO:53)

The vector containing the insert is then digested with SpeI and EcoRI toremove the resident “tag” sequence which can be replaced by thepoly-His/termination sequence. The produced V_(HH) can equally bedetected by using antibodies raised against the dromedary V_(HH)regions. Under laboratory conditions, V_(HH) regions are produced in theImmuno PBS vector in mg amounts per liter.

The invention also relates to a DNA library composed of nucleotidesequences coding for a heavy-chain immunoglobulin, such as obtained fromcells with rearranged immunoglobulin genes.

In a preferred embodiment of the invention, the library is prepared fromcells from an animal previously immunized against a determined antigen.This allows the selection of antibodies having a preselected specificityfor the antigen used for immunization.

In another embodiment of the invention, the amplification of the cDNA isnot performed prior to the cloning of the cDNA.

The heavy-chain of the four-chain immunoglobulins remains sequestered inthe cell by a chaperon protein (BIP) until it has combined with a lightchain. The binding site for the chaperon protein is the C_(H) 1 domain.As this domain is absent from the heavy chain immunoglobulins, theirsecretion is independent of the presence of the BIP protein or of thelight chain. Moreover the inventors have shown that the obtainedimmunoglobulins are not sticky and accordingly will not abnormallyaggregate.

The invention also relates to a process for the preparation of amonoclonal antibody directed against a determined antigen, the antigenbinding site of the antibody consisting of heavy polypeptide chains andwhich antibody is further devoid of light polypeptide chains, whichprocess comprises:

-   -   immortalizing lymphocytes, obtained for example from the        peripheral blood of Camelids previously immunized with a        determined antigen, with an immortal cell and preferably with        myeloma cells, in order to form a hybridoma,    -   culturing the immortalized cells (hybridoma) formed and        recovering the cells producing the antibodies having the desired        specificity.

The preparation of antibodies can also be performed without a previousimmunization of Camelids.

According to another process for the preparation of antibodies, therecourse to the technique of the hybridoma cell is not required.

According to such process, antibodies are prepared in vitro and they canbe obtained by a process comprising the steps of:

-   -   cloning into vectors, especially into phages and more        particularly filamentous bacteriophages, DNA or cDNA sequences        obtained from lymphocytes especially PBLs of Camelids previously        immunized with determined antigens,    -   transforming prokaryotic cells with the above vectors in        conditions allowing the production of the antibodies,    -   selecting the antibodies for their heavy-chain structure and        further by subjecting them to antigen-affinity selection,    -   recovering the antibodies having the desired specificity.

In another embodiment of the invention the cloning is performed invectors, especially into plasmids coding for bacterial membraneproteins. Procaryotic cells are then transformed with the above vectorsin conditions allowing the expression of antibodies in their membrane.

The positive cells are further selected by antigen affinity selection.

The heavy chain antibodies which do not contain the C_(H) 1 domainpresent a distinct advantage in this respect. Indeed, the C_(H) 1 domainbinds to BIP type chaperone proteins present within eukaryotic vectorsand the heavy chains are not transported out of the endocytoplasmicreticulum unless light chains are present. This means that in eukaryoticcells, efficient cloning of 4-chain immunoglobulins in non mammaliancells such as yeast cells can depend on the properties of the residentBIP type chaperone and can hence be very difficult to achieve. In thisrespect the heavy chain antibodies of the invention which lack the CH₁domain present a distinctive advantage.

In a preferred embodiment of the invention the cloning can be performedin yeast either for the production of antibodies or for the modificationof the metabolism of the yeast. As example, Yep 52 vector can be used.This vector has the origin of replication (ORI) 2 μ of the yeasttogether with a selection marker Leu 2.

The cloned gene is under the control of gall promoter and accordingly isinducible by galactose. Moreover, the expression can be repressed byglucose which allows the obtention of very high concentration of cellsbefore the induction.

The cloning between BamHI and SaII sites using the same strategy ofproduction of genes by PCR as the one described above, allows thecloning of camelid immunoglobulin genes in E. coli. As example ofmetabolic modulation which can be obtained by antibodies and proposedfor the yeast, one can site the cloning of antibodies directed againstcyclins, that is proteins involved in the regulation of the cellularcycle of the yeast (TIBS 16 430 J. D. Mc Kinney, N. Heintz 1991).Another example is the introduction by genetic engineering of anantibody directed against CD₂₈, which antibody would be inducible (forinstance by gall), within the genome of the yeast. The CD₂₈ is involvedat the level of the initiation of cell division, and therefore theexpression of antibodies against this molecule would allow an efficientcontrol of multiplication of the cells and the optimization of methodsfor the production in bioreactors or by means of immobilized cells.

In yet another embodiment of the invention, the cloning vector is aplasmid or a eukaryotic virus vector and the cells to be transformed areeukaryotic cells, especially yeast cells, mammalian cells for exampleCHO cells or simian cells such as Vero cells, insect cells, plant cells,or protozoan cells.

For more details concerning the procedure to be applied in such a case,reference is made to the publication of Marks et al, J. Mol. Biol. 1991,222:581-597.

Furthermore, starting from the immunoglobulins of the invention, or fromfragments thereof, new immunoglobulins or derivatives can be prepared.

Accordingly immunoglobulins replying to the above given definitions canbe prepared against determined antigens. Especially the inventionprovides monoclonal or polyclonal antibodies devoid of light polypeptidechains or antisera containing such antibodies and directed againstdetermined antigens and for example against antigens of pathologicalagents such as bacteria, viruses or parasites. As example of antigens orantigenic determinants against which antibodies could be prepared, onecan cite the envelope glycoproteins of viruses or peptides thereof, suchas the external envelope glycoprotein of a HIV virus, the surfaceantigen of the hepatitis B virus.

Immunoglobulins of the invention can also be directed against a protein,hapten, carbohydrate or nucleic acid.

Particular antibodies according to the invention are directed againstthe galactosyl α-1-3-galactose epitope.

The immunoglobulins of the invention allow further the preparation ofcombined products such as the combination of the heavy-chainimmunoglobulin or a fragment thereof with a toxin, an enzyme, a drug, ahormone.

As example one can prepare the combination of a heavy-chainimmunoglobulin bearing an antigen binding site recognizing a myelomaimmunoglobulin epitope with the abrin or mistletoe lectin toxin. Such aconstruct would have its uses in patient specific therapy.

Another advantageous combination is that one can prepare between aheavy-chain immunoglobulins recognizing an insect gut antigen with atoxin specific for insects such as the toxins of the different serotypesof Bacillus thuringiensis or Bacillus sphaericus. Such a constructcloned into plants can be used to increase the specificity or the hostrange of existing bacterial toxins.

The invention also proposes antibodies having different specificities oneach heavy polypeptide chains. These multifunctional, especiallybifunctional antibodies could be prepared by combining two heavy chainsof immunoglobulins of the invention or one heavy chain of animmunoglobulin of the invention with a fragment of a four-chain modelimmunoglobulin.

The invention also provides hetero-specific antibodies which can be usedfor the targetting of drugs or any biological substance like hormones.In particular they can be used to selectively target hormones orcytokines to a limited category of cells. Examples are a combination ofa murine or human antibody raised against interleukin 2 (IL₂) and aheavy-chain antibody raised against CD₄ cells. This could be used toreactivate CD₄ cells which have lost their IL₂ receptor.

The heavy-chain immunoglobulins of the invention can also be used forthe preparation of hetero-specific antibodies. These can be achievedeither according to the above described method by reduction of thebridges between the different chains and reoxydation, according to theusual techniques, of two antibodies having different specificities, butit can also be achieved by serial cloning of two antibodies for instancein the Immuno pBS vector.

In such a case, a first gene corresponding to the V_(HH) domaincomprised between Xho site and a Spe site is prepared as describedabove. A second gene is then prepared through an analogous way by usingas 5′ extremity a primer containing a Spe site, and as 3′ extremity aprimer containing a termination codon and an EcoRI site. The vector isthen digested with EcoRI and XhoI and further both V_(HH) genes aredigested respectively by Xho/Spe and by Spe/EcoRI.

After ligation, both immunoglobulin genes are serially cloned. Thespacing between both genes can be increased by the introduction ofaddition codons within the 5′ SpeI primer.

In a particular embodiment of the invention, the hinge region of IgG2immunoglobulins according to the invention is semi-rigid and is thusappropriate for coupling proteins. In such an application proteins orpeptides can be linked to various substances, especially to ligandsthrough the hinge region used as spacer. Advantageously the fragmentcomprises at least 6 amino acids.

According to the invention it is interesting to use a sequencecomprising a repeated sequence Pro-X, X being any amino-acid andpreferably Gln, Lys or Glu, especially a fragment composed of at least a3-fold repeat and preferably of a 12-fold repeat, for coupling proteinsto ligand, or for assembling different protein domains.

The hinge region or a fragment thereof can also be used for couplingproteins to ligands or for assembling different protein domains.

Usual techniques for the coupling are appropriate and especiallyreference may be made to the technique of protein engineering byassembling cloned sequences.

The antibodies according to this invention could be used as reagents forthe diagnosis in vitro or by imaging techniques. The immunoglobulins ofthe invention could be labelled with radio-isotopes, chemical orenzymatic markers or chemiluminescent markers.

As example and especially in the case of detection or observation withthe immunoglobulins by imaging techniques, a label like technetium,especially technitium 99 is advantageous. This label can be used fordirect labelling by a coupling procedure with the immunoglobulins orfragments thereof or for indirect labelling after a step of preparationof a complex with the technitium.

Other interesting radioactive labels are for instance indium andespecially indium 111, or iodine, especially I¹³¹, I¹²⁵ and I¹²³.

For the description of these techniques reference is made to the FRpatent application published under number 2649488.

In these applications the small size of the V_(HH) fragment is adefinitive advantage for penetration into tissue.

The invention also concerns monoclonal antibodies reacting withanti-idiotypes of the above-described antibodies.

The invention also concerns cells or organisms in which heavy-chainimmunoglobulins have been cloned. Such cells or organisms can be usedfor the purpose of producing heavy-chain immunoglobulins having adesired preselected specificity, or corresponding to a particularrepertoire. They can also be produced for the purpose of modifying themetabolism of the cell which expresses them. In the case of modificationof the metabolism of cells transformed with the sequences coding forheavy-chain immunoglobulins, these produced heavy-chain immunoglobulinsare used like antisense DNA. Antisense DNA is usually involved inblocking the expression of certain genes such as for instance thevariable surface antigen of trypanosomes or other pathogens. Likewise,the production or the activity of certain proteins or enzymes could beinhibited by expressing antibodies against this protein or enzyme withinthe same cell.

The invention also relates to a modified 4-chain immunoglobulin orfragments thereof, the V_(H) regions of which has been partialy replacedby specific sequences or amino acids of heavy chain immunoglobulins,especially by sequences of the V_(HH) domain. A particular modifiedV_(H) domain of a four-chain immunoglobulin, is characterized in thatthe leucine, proline or glutamine in position 45 of the V_(H) regionshas been replaced by other amino acids and preferably by arginine,glutamic acid or cysteine.

A further modified V_(H) or V_(L) domain of a four-chain immunoglobulin,is characterized by linking of CDR loops together or to FW regions bythe introduction of paired cysteines, the CDR region being selectedbetween the CDR₁ and the CDR₃, the FW region being the FW₂ region, andespecially in which one of the cysteines introduced is in position 31,33 of the FR₂ or 45 of CDR₂; and the other in CDR₃.

Especially the introduction of paired cysteines is such that the CDR₃loop is linked to the FW2 or CDR1 domain and more especially thecysteine of the CDR3 of the V_(H) is linked to a cysteine in position 31or 33 of the FR₂ or in position 45 of CDR₂.

In another embodiment of the invention, plant cells can be modified bythe heavy-chain immunoglobulins according to the invention, in orderthat they acquire new properties or increased properties.

The heavy-chain immunoglobulins of the invention can be used for genetherapy of cancer for instance by using antibodies directed againstproteins present on the tumor cells.

In such a case, the expression of one or two V_(HH) genes can beobtained by using vectors derived from parvo or adeno viruses. The parvoviruses are characterized by the fact that they are devoid ofpathogenicity or almost not pathogenic for normal human cells and by thefact that they are capable of easily multiplying in cancer cells (RusselS. J. 1990, Immunol. Today II. 196-200).

The heavy-chain immunoglobulins are for instance cloned withinHindIII/XbaI sites of the infectious plasmid of the murine MVM virus(pMM984). (Merchlinsky et al, 1983, J. Virol. 47, 227-232) and thenplaced under the control of the MVM38 promoter.

The gene of the V_(HH) domain is amplified by PCR by using a 5′ primercontaining an initiation codon and a HindIII site, the 3′ primercontaining a termination codon and a XbaI site.

This construct is then inserted between positions 2650 (HindIII) and4067 (XbaI) within the plasmid.

The efficiency of the cloning can be checked by transfection. The vectorcontaining the antibody is then introduced in permissive cells (NB-E) bytransfection.

The cells are recovered after two days and the presence of V_(HH)regions is determined with an ELISA assay by using rabbit antiserumreacting with the V_(HH) part.

The invention further allows the preparation of catalytic antibodiesthrough different ways. The production of antibodies directed againstcomponents mimicking activated states of substrates (as example vanadateas component mimicking the activated state of phosphate in order toproduce their phosphoesterase activities, phosphonate as compoundmimicking the peptidic binding in order to produce proteases) permits toobtain antibodies having a catalytic function. Another way to obtainsuch antibodies consists in performing a random mutagenesis in clones ofantibodies for example by PCR, in introducing abnormal bases during theamplification of clones. These amplified fragments obtained by PCR arethen introduced within an appropriate vector for cloning. Theirexpression at the surface of the bacteria permits the detection by thesubstrate of clones having the enzymatic activity. These two approachescan of course be combined. Finally, on the basis of the data availableon the structure, for example the data obtained by XRay crystallographyor NMR, the modifications can be directed. These modifications can beperformed by usual techniques of genetic engineering or by completesynthesis. One advantage of the V_(HH) of the heavy chainimmunoglobulins of the invention is the fact that they are sufficientlysoluble.

The heavy chain immunoglobulins of the invention can further be producedin plant cells, especially in transgenics plants. As example the heavychain immunoglobulins can be produced in plants using the pMon530plasmid (Roger et al. Meth Enzym 153 1566 1987) constitutive plantexpression vector as has been described for classical four chainantibodies (Hiat et al. Nature 342 76-78, 1989) once again using theappropriate PCR primers as described above, to generate a DNA fragmentin the right phase.

Other advantages and characteristics of the invention will becomeapparent in the examples and figures which follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Characterisation and purification of camel IgG by affinitychromatography on Protein A and Protein G SEPHAROSE (Pharmacia)

(A) shows, after reduction, the SDS-PAGE protein profile of the adsorbedand non adsorbed fractions of Camelus dromedarius serum. The fractionadsorbed on Protein A and eluted with NaCl 0.15 M acetic acid 0.58% showupon reduction (lane c) three heavy chain components of respectively 50,46 and 43 Kd and light chain (rabbit IgG in lane a). The fractionsadsorbed on a Protein G SEPHAROSE (Pharmacia) derivative which has beenengineered to delete the albumin binding region (lane e) and eluted with0.1 M gly HCl pH 2.7 lacks the 46 Kd heavy chain which is recovered inthe non adsorbed fraction (lane f). None of these components are presentin the fraction non adsorbed on Protein A (lane d), lane b contains themolecular weight markers.

(B) and (C) By differential elution, immunoglobulin fractions containingthe 50 and 43 Kd heavy chain can be separated. 5 ml of C. dromadariusserum is adsorbed onto a 5 ml Protein G SEPHAROSE column and the columnis extensively washed with 20 mM phosphate buffer, pH 7.0. Upon elutionwith pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid) a 100 Kd componentis eluted which upon reduction yields a 43 Kd heavy chain, (lane 1).After column eluant absorbance has fallen to background level a secondimmunoglobulin component of 170 Kd can be eluted with pH 2.7 buffer (0.1M glycine HC). This fraction upon reduction yields a 50 Kd heavy chainand a board light chain band (lane 2).

The fraction non adsorbed on Protein G is then brought on a 5 ml ProteinA SEPHAROSE column. After washing and elution with pH 3.5 buffer (0.15 MNaCl, 0.58% acetic acid) a third immunoglobulin of 100 Kd is obtainedwhich consists solely of 46 Kd heavy chains (lane 3).

FIG. 2: Immunoglobulins of Camelus bactrianus, Lama vicugna, Lama glamaand Lama pacos to Protein A (A lanes) and to Protein G (G lanes)analyzed on SDS-PAGE before FIG. (A) and after reduction FIG. (B)

10 μl of serum obtained from the different species were added toEPPENDORF® tubes containing 10 mg of Protein A or Protein G SEPHAROSEsuspended in 400 μl of pH 8.3 immunoprecipitation buffer (NaCl 0.2. M,Tris 0.01 M; EDTA 0.01 M, Triton X100 1%, ovalbumin 0.1%). The tubeswere slowly rotated for 2 hours at 4° C. After centrifugation thepellets were washed 3 times in buffer and once in buffer in which theTriton and ovalbumin had been omitted. The pellets were then resuspendedin the SDS-PAGE sample solution 70 μl per pellet with or withoutdithiotreitol as reductant. After boiling for 3 min at 100° C., thetubes were centrifuged and the supernatants analysed.

In all species examined the unreduced fractions (A) contain in additionto molecules of approximately 170 Kd also smaller major components ofapproximately 100 Kd. In the reduced sample (B) the constituant heavyand light chains are detected. In all species a heavy chain component(marked by an asterisk *) is present in the material eluted from theProtein A but absent in the material eluted from the Protein G.

FIG. 3: IgG₁, IgG₂ and IgG₃ were prepared from serum obtained fromhealthy or Trypanosama evansi infected Camelus dromedarius (CATT titer1/160 (3) and analysed by radioimmunopreci-pitation or Western Blottingfor anti trypanosome activity.

(A) ³⁵S methionine labelled Trypanosome evansi antigens lysate (500,000counts) was added to EPPENDORF tubes containing 10 μl of serum or, 20 μgof IgG₁, IgG₂ or IgG₃ in 200 μl of pH 8.3 immunoprecipitation buffercontaining 0.1 M TLCK as proteinase inhibitor and slowly rotated at 4°C. during one hour. The tubes were then supplemented with 10 mg ofProtein A SEPHAROSE suspended in 200 μl of the same pH 8.3 buffer andincubated at 4° C. for an additional hour.

After washing and centrifugation at 15000 rpm for 12 s, each pellet wasresuspended in 75 μl SDS-PAGE sample solution containing DTT and heatedfor 3 min. at 100° C. After centrifugation in an EPPENDORF minifuge at15000 rpm for 30 s, 5 μl of the supernatant was saved for radioactivitydetermination and the reminder analysed by SDS-PAGE and fluorography.The counts/5 μl sample are inscribed on for each line. (B) and (C) 20 μgof IgG₁, IgG₂ and IgG₃ from healthy and trypanosome infected animalswere separated by SDS-PAGE without prior reduction or heating. Theseparated samples were then electro transferred to a nitrocellulosemembrane, one part of the membrane was stained with Ponceau Red tolocalise the protein material and the reminder incubated with 1%ovalbumin in TST buffer (Tris 10 mM, NaCl 150 mM, Tween 0.05%) to blockprotein binding sites.

After blocking, the membrane was extensively washed with TST buffer andincubated for 2 hours with ³⁵S-labelled trypanosome antigen. Afterextensive washing, the membrane was dried and analysed byautoradiography. To avoid background and unspecific binding, thelabelled trypanosome lysate was filtered through a 45 μ millipore filterand incubated with healthy camel immunoglobulin and ovalbumin adsorbedon a nitrocellulose membrane.

FIG. 4: Purified IgG3 of the camel, by affinity chromatography onProtein A SEPHAROSE are partially digested with papain and separated onProtein A SEPHAROSE.

14 mg of purified IgG3 were dissolved in 0.1M phosphate buffer pH 7.0containing 2 mM EDTA. They were digested by 1 hour incubation at 37° C.with mercurypapain (1% enzyme to protein ratio) activated by 5.10⁴ Mcysteine. The digestion was blocked by the addition of excessiodoacetamide (4.10² M) (13). After centrifugation of the digest in anependorf centrifuge for 5 min at 15000 rpm, the papain fragments wereseparated on a protein A SEPHAROSE column into binding (B) and nonbinding (NB) fractions. The binding fraction was eluted from the columnwith 0.1M glycine HCl buffer pH 1.7.

FIG. 5: Schematic presentation of a model for IgG3 molecules devoid oflight chains.

FIG. 6: Schematic representation of immunoglobulins having heavypolypeptide chains and devoid of light chains, regarding conventionalfour-chain model immunoglobulin.

Representation of a hinge region.

FIG. 7: Alignement of 17 V_(HH) DNA sequences of Camel heavy chainimmunoglobulins (SEQ ID NOS: 92-108).

FIG. 8: Expression and purification of the camel V_(HH)21 protein fromE. coli

I HEAVY CHAIN ANTIBODIES IN CAMELIDS

When Camelus dromedarius serum is adsorbed on Protein G SEPHAROSE, anappreciable amount (25-35%) of immunoglobulins (Ig) remains in solutionwhich can then be recovered by affinity chromatography on Protein ASEPHAROSE (FIG. 1A). The fraction adsorbed on Protein G can bedifferentially eluted into a tightly bound fraction (25%) consisting ofmolecules of an unreduced apparent molecular weight (MW) of 170 Kd and amore weakly bound fraction (30-45%) having an apparent molecular weightof 100 Kd (FIG. 1B). The 170 Kd component when reduced yields 50 Kdheavy chains and large 30 Kd light chains. The 100 Kd fraction istotally devoid of light chains and appears to be solely composed ofheavy chains which after reduction have on apparent MW of 43 Kd (FIG.1C). The fraction which does not bind to Protein G can be affinitypurified and eluted from a Protein A column as a second 100 Kd componentwhich after reduction appears to be composed solely of 46 Kd heavychains.

The heavy chain immunoglobulins devoid of light chains total up to 75%of the molecules binding to Protein A.

As all three immunoglobulins bind to Protein A we refer to them as IgG:namely IgG₁ (light chain and heavy chain γ1 (50 Kd) binding to ProteinG, IgG₂ (heavy chain γ2 (46 Kd) non binding to Protein G and IgG₃ (heavychain γ3 (43 Kd) binding to Protein G. There is a possibility that thesethree sub(classes) can be further subdivided.

A comparative study of old world camelids (Camelus bactrianus andCamelus dromedarius) and new world camelids (lama pacos, lama glama,lama vicugna) showed that heavy chain immunoglobulins are found in allspecies examined, albeit with minor differences in apparent molecularweight and proportion. The new world camelids differs from the old worldcamelids in having a larger IgG₃ molecule (heavy chain immunoglobulinbinding to Protein G) in which the constituant heavy chains have anapparent molecular weight of 47 Kd (FIG. 2).

The abundance of the heavy chain immunoglobulins in the serum ofcamelids raises the question of what their role is in the immuneresponse and in particular whether they bear antigen binding specificityand if so how extensive is the repertoire. This question could beanswered by examining the immunoglobulins from Trypanosoma evansiinfected camels (Camelus dromedarius).

For this purpose, the corresponding fractions of IgG₁, IgG₂, IgG₃ wereprepared from the serum of a healthy camel and from the serum of camelswith a high antitrypanosome titer, measured by the Card AgglutinationTest (3). In radio-immunoprecipitation, IgG₁, IgG₂ and IgG₃ derived frominfected camel indicating extensive repertoire heterogeneity andcomplexity (FIG. 3A) were shown to bind a large number of antigenspresent in a ³⁵S methionine labelled trypanosome lysate.

In blotting experiments ³⁵S methionine labelled trypanosome lysate bindsto SDS PAGE separated IgG₁, IgG₂ and IgG₃ obtained from infected animals(FIG. 3B).

This leads us to conclude that the camelid heavy chain IgG₂ and IgG₃ arebona fide antigen binding antibodies.

An immunological paradigm states that an extensive antibody repertoireis generated by the combination of the light and heavy chain variable Vregion repertoires (6). The heavy chain immunoglobulins of the camelseem to contradict this paradigm.

Immunoglobulins are characterized by a complex I.E.F. (isoelectricfocussing) pattern reflecting their extreme heterogeneity. To determinewhether the two heavy chains constituting the IgG₂ and IgG₃ areidentical or not, the isoelectric focussing (I.E.F.) pattern wereobserved before and after chain separation by reduction and alkylationusing iodoacetamide as alkylating agent.

As this alkylating agent does not introduce additional charges in themolecule, the monomers resulting from the reduction and alkylation of aheavy chain homodimer will have practically the same isolectric point asthe dimer, whereas if they are derived from a heavy chain heterodimer,the monomers will in most cases differ sufficiently in isoelectric pointto generate a different pattern in I.E.F.

Upon reduction, and alkylation by iodoacetamide the observed pattern isnot modified for the Camelus dromedarius IgG₂ and IgG₃ indicating thatthese molecules are each composed of two identical heavy chains whichmigrate to the same position as the unreduced molecule they originatedfrom.

In contrast, the I.E.F. pattern of IgG₁ is completely modified afterreduction as the isoelectric point of each molecule is determined by thecombination of the isoelectric points of the light and heavy chainswhich after separation will each migrate to a different position.

These findings indicate that the heavy chains alone can generate anextensive repertoire and question the contribution of the light chain tothe useful antibody repertoire. If this necessity be negated, what otherrole does the light chain play.

Normally, isolated heavy chain from mammalian immunoglobulins tend toaggregate considerably but are only solubilized by light chains (8, 9)which bind to the C_(H) 1 domain of the heavy chain.

In humans and in mice a number of spontaneous or induced myelomasproduce a pathological immunoglobulin solely composed of heavy chains(heavy chain disease). These myeloma protein heavy chains carrydeletions in the C_(H) 1 and V_(HH) domains (10). The reason why fulllength heavy chains do not give rise to secreted heavy chain in suchpathological immunoglobulins seems to stem from the fact that thesynthesis of Ig involves a chaperoning protein, the immunoglobulin heavychain binding protein or BIP (11), which normally is replaced by thelight chain (12). It is possible that the primordial role of the lightchain in the four-chain model immunoglobulins is that of a committedheavy chain chaperon and that the emergence of light chain repertoireshas just been an evolutionary bonus.

The camelid γ2 and γ3 chains are considerably shorter than the normalmammalian γ chain. This would suggest that deletions have occurred inthe C_(H) 1 domain. Differences in sizes of the γ2 and γ3immunoglobulins of old and new world camelids suggests that deletionsoccurred in several evolutionary steps especially in the C_(H) 1 domain.

II THE HEAVY CHAIN IMMUNOGLOBULINS OF THE CAMELIDS LACK THE C_(H)1DOMAIN

The strategy followed for investigating the heavy chain immunoglobulinprimary structure is a combination of protein and cDNA sequencing; theprotein sequencing is necessary to identify sequence strechescharacteristic of each immunoglobulin. The N-terminal of theimmunoglobulin being derived from the heavy chain variable regionrepertoire only yields information on the V_(HH) subgroups (variableregion of the heavy chain) and cannot be used for class or subclassidentification. This means that sequence data had to be obtained frominternal enzymatic or chemical cleavage sites.

A combination of papain digestion and Protein A affinity chromatographyallowed the separation of various fragments yielding information on thegeneral structure of IgG3.

The IgG3 of the camel (Camelus dromedarius) purified by affinitychromatography on Protein A SEPHAROSE were partially digested withpapain and the digest was separated on Protein A SEPHAROSE into bindingand non binding fractions. These fractions were analysed by SDS PAGEunder reducing and non reducing conditions (FIG. 4).

The bound fraction contained two components, one of 28 Kd and one of14.4 Kd, in addition to uncleaved or partially cleaved material. Theywere well separated by gel electrophoresis (from preparative 19%SDS-PAGE gels ) under non reducing conditions and were further purifiedby electroelution (in 50 nM amonium bicarbonate, 0.1% (w/v) SDS using aBioRad electro-eluter). After lyophilization of these electroelutedfractions, the remaining SDS was eliminated by precipitating the proteinby the addition of 90% ethanol, mixing and incubating the mixtureovernight at −20° C. (14). The precipitated protein was collected in apellet by centrifuging (15000 rpm, 5 min) and was used for proteinsequencing. N-terminal sequencing was performed using the automatedEdman chemistry of an Applied Biosystem 477A pulsed liquid proteinsequencer. Amino acids were identified as their phenylthiohydantoin(PTH) derivatives using an Applied Biosystem 120 PTH analyser. Allchemical and reagents were purchased from Applied Biosystems. Analysisof the chromatographic data was performed using Applied Biosystemssoftware version 1.61. In every case the computer aided sequenceanalysis was cofirmed by direct inspection of the chromatograms from thePTH analyser. Samples for protein sequencing were dissolved in either50% (v/v) trifluoroacetic acid(TFA) (28 Kd fragment) or 100% TFA (14 Kdfragment). Samples of dissolved protein equivalent to 2000 pmol (28 Kdfragment) or 500 pmol (14 Kd fragment) were applied to TFA-treated glassfibre discs. The glass fibre discs were coated with BioBrene (3 mg) andprecycled once before use.

N-terminal sequencing of the 28 Kd fragment yields a sequence homologousto the N-terminal part of γ C_(H) 2 domain and hence to the N-terminalend of the Fc fragment. The N-terminal sequence of the 14.4 Kd fragmentcorresponds to the last lysine of a γ C_(H) 2 and the N-terminal end ofa γ C_(H) 3 domain (Table 1). The molecular weight (MW) of the papainfragments and the identification of their N-terminal sequences led us toconclude that the C_(H) 2 and C_(H) 3 domains of the γ3 heavy chains arenormal in size and that the deletion must occur either in the C_(H) 1 orin the V_(HH) domain to generate the shorted γ3 chain. The fractionswhich do not bind to Protein A SEPHAROSE contain two bands of 34 and 17Kd which are more diffuse is SDS PAGE indicating that they originatefrom the variable N-terminal part of the molecule (FIG. 4).

Upon reduction, a single diffuse band of 17 Kd is found indicating thatthe 34 Kd is a disulfide bonded dimer of the 17 Kd component. The 34 Kdfragment apparently contains the hinge and the N-terminal domain V_(HH).

The protein sequence data can be used to construct degenerateoligonucleotide primers allowing PCR amplification of cDNA or genomicDNA.

It has been shown that the cells from camel spleen imprint cells reactedwith rabbit and anti camel immunoglobulin sera and that the spleen washence a site of synthesis of at least one immunoglobulin class. cDNA wastherefore synthetised from camel spleen mRNA. The conditions for theisolation of RNA were the following: total RNA was isolated from thedromedary spleen by the guanidium isothiocyanate method (15). mRNA waspurified with oligo T-paramagnetic beads.

cDNA synthesis is obtained using 1 μg mRNA template, an oligodT primerand reverse transcriptase (BOERHINGER MAN). Second strand cDNA isobtained using RNAse H and E coli DNA polymerase I according to thecondition given by the supplier.

Relevant sequences were amplified by PCR: 5 ng of cDNA was amplified byPCR in a 100 μl reaction mixture (10 mM Tris-HCl pH 8.3, 50 mM KCl, 15mM MgCl₂, 0.01% (w/v) gelatine, 200 μM of each dNTP and 25 pmoles ofeach primer) overlaid with mineral oil (Sigma).

Degenerate primers containing EcoRI and KpnI sites and further clonedinto pUC 18. After a round of denaturing and annealing (94° C. for 5 minand 54° C. for 5 min), 2 units of Taq DNA polymerase were added to thereaction mixture before subjecting it to 35 cycles of amplification: 1min at 94° C. (denature) 1 min at 54° C. (anneal), 2 min at 72° C.(elongat amplify DNA sequences between V_(HH) and C_(H)2 domains, (#72clones), the PCR was performed in the same conditions with the exceptionthat the annealing temperature was increased to 60° C.

One clone examined (#56/36) had a sequence corresponding to theN-terminal part of a C_(H) 2 domain identical to the sequence of the 28Kd fragment. The availability of this sequence data allowed theconstruction of an exact 3′ primer and the cloning of the region betweenthe N-terminal end of the V_(HH) and the C_(H) 2 domain.

5′ primers corresponding to the mouse V_(HH) (16) and containing a XhoIrestriction site were used in conjunction with the 3′ primer in which aKpnI site had been inserted and the amplified sequences were cloned intopBLUESCRIPT®. Clone #56/36 which displayed two internal HaeIII sites wasdigested with this enzyme to produce a probe to identify PCR positiveclones.

After amplification the PCR products were checked on a 1.2% (w/v)agarose gel. Cleaning up of the PCR products included aphenol-chloroform extractio followed by further purification by HPLC(GEN-PAC FAX column, Waters) and finally by using the MERMAID orGENECLEAN II kit, BIO 101, Inc) as appropriate. After these purificationsteps, the amplified cDNA was then digested with EcoRI and KpnI forseries #56 clones and with XhoI and KpnI for series #72 clones. A finalphenol-chloroform extraction preceded the ligation into pUC 18 (series#56 clones) or into pBLUESCRIPT® (series #72 clones).

All the clones obtained were smaller that the 860 base pairs to beexpected if they possessed a complet V_(HH) and C_(H)1 region. Partialsequence data corresponding to the N-terminal of the V_(HH) regionreveals that out of 20 clones, 3 were identical and possibly notindependent. The sequences obtained ressemble the human subgroup III andthe murine subgroups IIIa and IIIb ( 2).

Clones corresponding to two different sets of C_(H) 2 protein sequenceswere obtained. A first set of sequences (#72/41) had a N-terminal C_(H)2 region identical to the one obtained by protein sequencing of the 28Kd papain fragments of the γ3 heavy chain, a short hinge regioncontaining 3 cysteines and a variable region corresponding to theframework (FR4) residues encoded by the J minigenes adjoining the hinge.The C_(H) 1 domain is entirely lacking. This cDNA corresponds to the γ3chain (Table 4).

In one closely related sequence (#72/1) the proline in position 259 isreplaced by threonine.

The sequence corresponding to the C_(H) 3 and the remaining part of theC_(H) 2 was obtained by PCR of the cDNA using as KpnI primer a poly T inwhich a KpnI restriction site had been inserted at the 5′ end. The totalsequence of the γ3 chain corresponds to a molecular weight (MW) which isin good agreement with the data obtained from SDS PAGE electrophoresis.

The sequence of this γ3 chain presents similarities with other γ chainsexcept that it lacks the C_(H) 1 domain, the V_(HH) domain beingadjacent to the hinge.

One or all three of the cysteines could be probably responsible forholding the two γ3 chains together.

These results have allowed us to define a model for the IgG3 moleculebased on sequence and papain cleavage (FIG. 5).

Papain can cleave the molecule on each side of the hinge disulfides andalso between C_(H) 2 and C_(H) 3. Under non reducing conditions theV_(HH) domains of IgG3 can be isolated as disulfide linked dimer or asmonomer depending on the site of papain cleavage.

A second set of clones #72/29 had a slightly different sequence for theC_(H) 2 and was characterized by a very long hinge immediately precededby the variable domain. This hinge region has 3 cysteines at itsC-terminal end in a sequence homologeous to the γ3 hinge. Such secondset of clones could represent the IgG2 subclass. For the constant partof the γ3 and also for the putative γ2, most clones are identicalshowing the γ2 or γ3 specific sequences. A few clones such as #72/1however show minor differences. For instance in the case of clones #72/1two nucleotide differences are detected.

Several V_(HH) regions cDNA's have now been totally or partiallysequenced with the exception of a short stretch at the N-terminal endwhich is primer derived.

Upon translation the majority shows the characteristic heavy chain Ser₂₁Cys₂₂ and Tyr₉₀ Tyr₉₁ Cys₉₂ sequences, of the intra V_(HH) regiondisulfide bridge linking residues 22 and 92. All these clones have asequence corresponding to the framework 4 (FR4) residues of the variableregion immediately preceding the postulated hinge sequence (Table 3).This sequence is generated by the J minigenes and is in the majority ofcases similar to the sequence encoded by the human and murine Jminigenes. The sequence length between region Cys₉₂ and the C-terminalend of the V_(HH) regions is variable and, in the sequences determined,range from 25 to 37 amino-acids as one might expect from therearrangements of J and D minigenes varying in length.

Several important questions are raised by the sole existence of theseheavy chain immunoglobulins in a non pathological situation. First ofall, are they bonafide antibodies? The heavy chain immunoglobulinsobtained from trypanosome infected camels react with a large number ofparasite antigens as shown in part I of these examples. This impliesthat the camelid immune system generates an extensive number of bindingsites composed of single V_(HH) domains. This is confirmed by thediversity of the V_(HH) regions of the heavy chain immunoglobulinsobtained by PCR.

The second question is “how are they secreted?”. The secretion ofimmunoglobulin heavy chains composing four-chain model immunoglobulinsdoes not occur under normal conditions. A chaperoning protein, the heavychain binding protein, or BIP protein, prevents heavy chains from beingsecreted. It is only when the light chain dispplaces the BIP protein inthe endoplasmatic reticulum that secretion can occur (13).

The heavy chain dimer found in the serum of human or mice with theso-called “heavy chain disease” lack the C_(H) 1 domains thought toharbour the BIP site (14). In the absence of thi domain the BIP proteincan no longer bind and prevent the transport of the heavy chains.

The presence in camels of a IgG1 class composed of heavy and lightchains making up between 25% and 50% of the total IgG molecules alsoraises the problem as to how maturation and class switching occurs andwhat the role of the light chain is. The camelid light chain appearsunusually large and heterogeneous when examined in SDS PAGE.

The largest dimension of an isolated domain is 40 Å and the maximumattainable span between binding sites of a conventional IgG with C_(H) 1and V_(HH) will be of the order of 160 Å (2 V_(HH)+2 C_(H) 1) (19). Thedeletion of C_(H) 1 domain in the two types of heavy chain antibodiesdevoid of light chains, already sequenced has, as a result, amodification of this maximum span (FIG. 6). In the IgG3 the extremedistance between the extremities of the V_(HH) regions will be of theorder of 80 Å (2V_(HH)). This could be a severe limitation foragglutinating or cross linking. In the IgG2 this is compensated by theextremely long stretch of hinge, composed of a 12-fold repeat of thesequence Pro-X (where X is Gln, Lys or Glu) and located N-terminal tothe hinge disulfide bridges. In contrast, in the human IgG3, the verylong hinge which also apparently arose as the result of sequenceduplication does not contribute to increase the distance spanning thetwo binding sites as this hinge is inter-spersed with disulfide bridges.

The single V_(HH) domain could also probably allow considerablyrotational freedom of the binding site versus the Fc domain.

Unlike myeloma heavy chains which result probably from C_(H) 1 deletionin a single antibody producing cell, or heavy chain antibodies producedby expression cloning (15); the camelid heavy chain antibodies (devoidof light chains) have emerged in a normal immunological environment andit is expected that they will have undergone the selective refinement inspecificity and affinity accompanying B cell maturation.

Expression and Purification of the Camel V_(HH)21 (DR21 on FIG. 7)Protein from E. coli

The clones can be expressed in several types of expression vectors. Asan example using a commercially available vector Immuno PBS (Huse et al:Science (1989) 246, 1275), clones produced in BLUESCRIPT® according tothe above described procedure, have been recovered by PCR using the sameXhoI containing 5′ primer and a new 3′ primer, corresponding to residues113-103 in the framework of the immunoglobulins, in which an Spe sitehas been constructed: TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ IDNO:51). This procedure allowed the cloning of the V_(HH) in the Xho/Spesite of the Immuno PBS vector. However, the 3′ end of the gene was notin phase with the identification “tag” and the stop codon of the vector.To achieve this, the construct was cut with Spe and the 4 base overhangswere filled in, using the Klenow fragment after which the vector wasreligated.

The expression vector plasmid ipBS (immunopBS) (Stratacyte) contains apel B leader sequence which is used for immunoglobulin chain expressionin E. coli under the promotor pLAC control, a ribosome binding site, andstop codons. In addition, it contains a sequence for a c-terminaldecapeptide tag.

E. coli JM101 harboring the ipBS-V_(HH) 21 plasmid was grown in 11 of TBmedium with 100 μg/ml ampicillin and 0.1% glucose at 32° C. Expressionwas induced by the addition of 1 mM IPTG (final concentration) at anOD₅₅₀ of 1.0. After overnight induction at 28° C., the cells wereharvested by centrifugation at 4,000 g for 10 min (4° C.) andresuspended in 10 ml TES buffer (0.2 M Tris-HCL pH 8.0, 0.5 mM EDTA, 0.5M sucrose). The suspension was kept on ice for 2 hours. Periplasmicproteins were removed by osmotic shock by addition of 20 ml TES bufferdiluted 1:4 v/v with water, kept on ice for one hour and subsequentlycentrifugated at 12,000 g for 30 min. at 4° C. The supernatantperiplasmic fraction was dialysed against Tris-HCl pH 8.8, NaCl 50 mM,applied on a fast Q SEPHAROSE flow (Pharmacia) column, washed with theabove buffer prior and eluted with a linear gradient of 50 mM to 1 MNaCl in buffer.

Fractions containing the V_(HH) protein were further purified on aSUPERDEX 75 column (Pharmacia) equilibrated with PBS buffer (0.01 Mphosphate pH 7.2, 0.15 M NaCl). The yield of purified V_(HH) proteinvaries from 2 to 5 mg/l cell culture.

Fractions were analyzed by SDS-PAGE(I). Positive identification of thecamel V_(HH) antibody fragment was done by Western Blot analysis usingantibody raised in rabbits against purified camel IgGH₃ and ananti-rabbit IgG-alkaline phosphatase conjugate (II).

As protein standards (Pharmacia) periplasmic proteins prepared from 1 mlof IPTG-induced JM101/ipBS V_(HH) 21 were used. FIG. 8 shows:C,D:fractions from fast S SEPHAROSE column chromatography (C:Eluted at650 mM NaCl D:Eluted at 700 mM NaCl) E,F:fractions from SUPERDEX 75column chromatography.

As can be seen, the major impurity is eliminated by ionexchangechromatography and the bulk of the remaining impurities are eliminatedby gel filtration. TABLE 1 Comparison of the N terminal Camel CH2 andCH3 sequences with the translated cDNA sequences of Camelimmunoglobulins and with the corresponding human, gamma, sequences.(Numbering according to Kabat et at (1987)(7).       250                 260                 270 Camel Y3 28Kd - L P GG P S V F V F P P K P K D V L S I X G X P - - SEQ ID 54 Clone # 72/1 - LP G G P S V F V F P T K P K D V L S I S G R P - - SEQ ID 55 Clone# 72/4 - L P G G P S V F V F P P K P K D V L S I S G R P - - SEQ ID 56Clone # 72/29 - L L G G P S V F I F P P K P K D V L S I S G R P - - SEQID 57 Human Y1Y3 - L L G G P S V F L F P P K P K D T L M I S R T P - -SEQ ID NO:112 C_(H)2 Y2 - V A - G P S V F L F P P K P K D T L M I S R TP - - SEQ ID NO:113 Y4 - F L G G P S V F L F P P K P K D T L M I S R TP - - SEQ ID NO:114 C_(H)2|C_(H)3 360                     370 Camel Yy314Kd - K|G Q T R E P Q V Y T L A P X R L E L - - SEQ ID NO:54 Human -K|G Q P R E P Q V Y T L P P S R D E L - - SEQ ID NO:115 C_(H)2/C_(H)3Y2, Y3 - K|G Q P R E P Q V Y T L P P S R E E M - - SEQ ID NO:116 Y4 -K|G Q P R E P Q V Y T L P P S Q E E M - - SEQ ID NO:117

TABLE 2 A comparison of N Terminal Fr 1 regions of Camel V_(HH) with aHuman V_(H)III subgroup protein and a mouse V_(H)IIIA subgroup protein.The invariable subgroup specific residues are grayed. Primer Derived                  10                  20                  30              G G S V Q T G G S L R L S C E T S G L T F D # 72/4 SEQ IDNO:1               G G S V Q T G G S L R L S C A V S G F S F S # 72/3SEQ ID NO:2               G G S E Q G G G S L R L S C A T S G Y T Y G# 72/7 SEQ ID NO:3               G G S V Q P G G S L T L S C T V S G A TY S # 72/17 SEQ ID NO:4               G G S V Q A G G S L R L S C T G SG F P Y S # 72/18 SEQ ID NO:5 D V Q L V A S G G G S V G A G G S L R L SC T A S G D S F S # 72/2 SEQ ID NO:58 E V K L V E S G G G L V E P G G SL R L S C A T S G F T F S Mouse V_(H)III_(A) SEQ ID NO:118   E V Q L L SG G C L V Q P G G S L R L S C A A S C F T F S Human V_(H)III SEQ IDNO:119

TABLE 3 Comparison of some Framework 4 residues found in the CamelV_(HH) region with the Framework 4 residues corresponding to theconsensus region of the Human and Mouse J minigenes. FrameWork 4 J GenesHuman W G Q G T L V T V S S SEQ ID NO:9 J1, J4, J5 W G R G T L V T V S SSEQ ID NO:130 J2 W G Q G T T V T V S S SEQ ID NO:120 J6 W G Q G T M V TV S S SEQ ID NO:121 J3 Murine W G Q G T T L T V S S SEQ ID NO:122 J1 W GQ G T L V T V S S SEQ ID NO:9 J2 W G Q G T S V T V S S SEQ ID NO:123 J3W G A G T T V T V S S SEQ ID NO:124 J4 cDNA Clones Camel W G Q G T Q V TV S S SEQ ID NO:8 Clones W G Q G T Q V T V S S SEQ ID NO:8 # 72/19 =# 72/3 W G Q G T L V T V S S SEQ ID NO:9 1 Clone W G R G T Q V T V S SSEQ ID NO:59 # 72/24 W G Q G T H V T V S S SEQ ID NO:60 # 72/21 W G Q GI Q V T A S S SEQ ID NO:61 # 72/16

TABLE 4

TABLE 5

REFERENCES

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Issue n* 27.

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1-17. (canceled)
 18. Immunoglobulin having determined antigenspecificity, and having a variable fragment which is derived from aso-called heavy-chain immunoglobulin having two heavy polypeptide chainscapable of recognizing and binding one or several antigens and which isfurther naturally devoid of light chains, wherein the immunoglobulinhaving determined antigen specificity is devoid of C_(H)1 constantregion between the variable region and the hinge region and has withinits constant region at least part of a constant region of a humanantibody.
 19. Immunoglobulin according to claim 18 wherein the variablefragment is derived from a camelid heavy-chain immunoglobulin. 20.Immunoglobulin according to claim 19, wherein the variable fragment is avariable fragment of a camelid heavy-chain immunoglobulin. 21.Immunoglobulin according to claim 18 wherein the constant regioncomprises a CH2 domain or a CH3 domain or, both CH2 and CH3 domains, ofa human antibody.
 22. Immunoglobulin according to claim 19, wherein theconstant region comprises a CH2 domain or a CH3 domain or, both CH2 andCH3 domains, of a human antibody.
 23. Immunoglobulin according to claim18, which is derived from a heavy-chain immunoglobulin naturally devoidof light chains and having two heavy polypeptide chains capable ofrecognizing and binding one or several antigens.
 24. Immunoglobulinaccording to claim 18, which is derived from a heavy-chainimmunoglobulin naturally devoid of light chains and having two heavypolypeptide chains capable of binding and recognizing one or severalantigens, being humanized by replacement of part of its variablesequence, by part of a human antibody.
 25. Immunoglobulin according toclaim 24 wherein the variable sequence is humanized by replacement ofone or more of its framework residues, by human framework residues. 26.Immunoglobulin according to claim 23 wherein the heavy-chainimmunoglobulin naturally devoid of light chains is a camelidimmunoglobulin.
 27. Immunoglobulin according to claim 24 wherein theheavy-chain immunoglobulin naturally devoid of light chains is a camelidimmunoglobulin.